Abstract

Enzyme kinetics of GTI-2040 (5′-GGC TAA ATC GCT CCA CCA AG-3′), a phosphorothioate ribonucleotide reductase antisense, were investigated for the first time in 3′ exonuclease solution and human liver microsomes (HLMs), using the ion-pair high-performance liquid chromatogram method for quantification of the parent drug and two major 3′N-1 and 3′N-2 metabolites. Enzyme kinetics of GTI-2040 in 3′-exonuclease solution were found to be well characterized by the Michaelis-Menten model, using the sum of formation rates of 3′N-1 and 3′N-2 (∼total metabolism) because of sequential metabolism. In HLMs, a biphasic binding was observed for GTI-2040 with high- and low-affinity constants (Kds) of 0.03 and 3.8 μM, respectively. Enzyme kinetics of GTI-2040 in HLMs were found to deviate from Michaelis-Menten kinetics when the total GTI-2040 substrate was used. However, after correction for the unbound fractions, the formation rate of total metabolites could be described by Michaelis-Menten kinetics. Using the free substrate fraction, the Km and Vmax of GTI-2040 were determined to be 6.33 ± 3.2 μM and 16.5 ± 8.4 nmol/mg/h, respectively. Using these values, in vitro hepatic intrinsic clearance (CLint) in HLM was estimated to be 2.61 ± 0.56 ml/h. The CLint was then used to predict GTI-2040's in vivo intrinsic clearance in humans by a microsomal protein scaling factor, which gave a mean value of 182.7 l/h, representing 24.1% of the observed in vivo mean scaled hepatic intrinsic clearance of 758.7 l/h in patients with acute myeloid leukemia. We concluded that the saturable nonspecific binding of GTI-2040 in HLMs complicated the interpretation of its enzyme kinetics, and scaled intrinsic clearance from HLMs only partially predicted the in vivo intrinsic clearance.

Antisense oligonucleotides (ODNs) are short, single-strand DNA molecules designed to hybridize with specific mRNA strands, thereby selectively inhibiting the production of specific gene products (Stein and Cheng, 1993; Dias and Stein, 2002). This approach has been explored to target expression of genes important for malignant transformation and other pathogenetic mechanisms. For a successful therapeutic use of ODN compounds robust in vivo, stability is critical. Previously used unmodified ODNs degraded rapidly in biological fluids by nucleases, which limited their clinical use. More recently, by substituting one of the nonbridge oxygen atoms, phosphorothioate analogs have been synthesized and shown to be more resistant to exonucleases than the unmodified ODNs (Crooke, 2001; Aboul-Fadl, 2005). Several phosphorothioate ODNs (PS-ODNs) are currently undergoing clinical evaluation for a number of diseases, including cancer, viral infections, and inflammatory disorders (Crooke, 2001; Jansen and Zangemeister-Wittke, 2002; Marcucci et al., 2005). It has been reported that the in vivo pharmacodynamic effects of antisense correlate with the intracellular drug levels and clinical response (Yu et al., 2001; Dai et al., 2005a; Marcucci et al., 2005). Moreover, it is anticipated that a well defined pharmacokinetics-pharmacodynamics correlation may help to improve the optimization of dose regimens of PS-ODNs. Because pharmacokinetics of PS-ODNs are largely driven by their disposition and metabolism (Crooke, 2001; Yu et al., 2004), it has become increasingly important to obtain a fundamental understanding of the metabolism and degradation kinetics of these compounds. Although a significant effort has been invested in identification of the in vivo and in vitro metabolites of PS-ODNs, and the 3′ end progressively deleted metabolites mediated by the 3′ exonuclease hydrolysis has been found to be the major pathway of metabolism for various PS-ODNs (Cohen et al., 1997; Crooke et al., 2000; Dai et al., 2005b; Wei et al., 2006a), few studies focusing on the enzyme kinetics of PS-ODNs have been reported. In addition, it has been reported that liver is the most important organ for metabolism and disposition of PS-ODNs, although kidneys, spleen, bone marrow, and lymph nodes metabolism also play a role (Cossum et al., 1993; Butler et al., 1997; Noll et al., 2005). Therefore, it is important to evaluate the contribution of hepatic metabolism to the clearance of PS-ODNs and to predict the in vivo CLint from in vitro metabolism.

GTI-2040 is a 20-mer phosphorothioate oligonucleotide that inhibits the production of the R2 subunit of ribonucleotide reductase, which is essential for DNA synthesis (Lee et al., 2003). GTI-2040 has been shown recently to have promising response and acceptable tolerability in phase I clinical trials as a single agent or in combination with cytarabine for the treatment of advanced solid tumors and acute myeloid leukemia (AML) (Desai et al., 2005; Klisovic et al., 2008). Using a highly specific ion-pair HPLC/tandem mass spectrometry method (Griffey et al., 1997; Gilar and Bouvier, 2000; Dai et al., 2005b), we recently reported the metabolism of GTI-2040 as the sequential nucleotide deletion via the 3′ exonuclease (Wei et al., 2006). A series of progressively 3′ chain short-end metabolites were identified in several biological matrices including plasma from AML patients, solutions containing 3′ exonuclease, and in human liver microsomes. Herein, we investigated the enzyme kinetics of GTI-2040 in a system containing either 3′ exonuclease or human liver microsomes (HLMs). Formation rates of the 3′ end metabolites were monitored and used to characterize the enzyme kinetics of GTI-2040. To evaluate the contribution of liver microsomes to the metabolism of GTI-2040, the in vitro intrinsic clearance in HLMs was determined. The in vitro CLint was extrapolated to a predicted in vivo value that was compared with the intrinsic clearance determined from patients.

Materials and Methods

Drugs and Chemicals. GTI-2040, a 20-mer phosphorothioate oligonucleotide with the sequence 5′-GGC TAA ATC GCT CCA CCA AG-3′, was provided by the National Cancer Institute (Bethesda, MD) and used without further purification. Putative 3′ end metabolites of GTI-2040, 3′N-1, 3′N-2, and 3′N-3-GTI-2040s (heretofore, GTI-2040 is omitted), from which one to three nucleotides were deleted from the 3′ end, respectively, and the internal standard (IS), PS-dC 28, a 28-mer polycytidine phosphorothioate oligonucleotide, were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). The purity and identity of all of the oligomers were verified by HPLC/UV/mass spectrometry (Finnigan LCQ; Thermo Fisher Scientific, Waltham, MA). Phosphodiesterase I (EC 3.1.4.1) from snake (Crotalus adamanteus) venom, a 3′- to 5′-exonuclease, was obtained from USB (Cleveland, OH). Pooled human liver microsomes from 18 individuals (10 male and eight female donors) were obtained as a stock emulsion (20 mg protein/ml in 250 mM sucrose) from BD Biosciences (San Jose, CA). HPLC-grade methanol, TEA (99.5%), triethylammonium bicarbonate (TEAB), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; 99.8%) were purchased from Sigma-Aldrich (St. Louis, MO). HPLC-grade water was generated by an E-pure water purification system (Barnstead, Dubuque, IA).

Sample Preparation Solid Phase Extraction. Samples containing GTI-2040 and its metabolites were thawed and centrifuged at 1000g for 5 min. One milliliter of the supernatant was mixed with 2 ml of 0.1 M TEAB, and the mixture was allowed to stand at room temperature for 30 min for ion-pair formation between the oligonucleotide and TEA. GTI-2040 and related metabolites were extracted with an Oasis HLB cartridge packed with 60 mg of material (Waters, Milford, MA). The extraction tubes were preconditioned with 1 ml of acetonitrile followed by 1 ml of 0.1 M TEAB, pH 8.0. Samples mixed with 0.1 M TEAB were loaded onto these preconditioned solid phase columns. The protein and salts were removed by sequential washings with 3 ml of 0.1 M TEAB, 3 ml of distilled water, and 3 ml of 10% acetonitrile in 0.1 M TEAB by gravity flow. Then, GTI-2040 and its related metabolites were eluted with 3 ml of 50% acetonitrile, and the eluant was evaporated to dryness under N2. The residue was reconstituted with 150 μl of mobile phase A, and a 50-μl aliquot was analyzed by HPLC.

HPLC Conditions. Previously reported HPLC conditions (Dai et al., 2005b; Wei et al., 2006a) were used for the separation of GTI-2040 and its chain-shortened metabolites. Briefly, the separation was achieved on a 2.5-μm Waters Xterra MS18 column (50 × 2.1 mm) coupled to an MS C18 10-× 2.1-mm guard column (Waters). The mobile phase was prepared as mobile phase A, consisting of 100 mM HFIP buffered to pH 8.3 with 8.4 mM TEA, and mobile phase B, consisting of 100 mM HFIP and 8.6 mM TEA, pH 8.3, in methanol [50:50 (v/v)]. Gradient elution was used for the oligomer separation at a flow rate of 0.2 ml/min. The elution was initiated with 30% mobile phase B, followed by a linear increase to 45% in 30 min, and returned to 30% in 2 min, which was maintained for 8 min before the next run. The column temperature was set to 50°C throughout the analysis using a column heater (Keystone, Waltham, MA). The autosampler temperature was kept at 4°C throughout the sample run. A Shimadzu HPLC system consisting of two LC-10AT vp pumps, a SIL-10AD vp autosampler, and a SPD-10A vp UV-VIS detector (Shimadzu, Kyoto, Japan) was used for quantification. The detected wavelength was set at 260 nm.

Quantitation. Quantitation of metabolites was determined by comparing the formed metabolites with the control GTI-2040 at each initial concentration in the kinetic studies. Peak area ratios of GTI-2040 or each formed metabolites to the IS in each HPLC chromatogram were determined. The concentrations of formed metabolites were then calculated using the following equation: where Cm is the concentration of the formed metabolites in the reaction medium, CG,I is the known concentration of control GTI-2040, Rm is the peak area ratio of metabolites to the IS, RG,I is the peak area ratio of control GTI-2040 to the IS, and EG and Em are the extinction coefficients of GTI-2040 and metabolites, respectively. Concentrations of two major metabolites, 3′N-1 and 3′N-2, were calculated according to eq. 1 with the EG/Em(3′N-1) ratio of 1.053 and the EG/Em(3′N-2) ratio of 1.128, respectively. The other metabolites were too low to be detected; thus, they were not considered.

Free Drug Concentration. The extent of binding of GTI-2040 to human liver microsomes was determined by ultrafiltration method at protein concentration of 0.2 mg/ml for HLM and GTI-2040 concentration ranging between 0.1 and 20 μM used in the kinetic studies. EDTA (5 mM) was added to inhibit the nuclease activity in microsomes. After incubation in a 37°C shaking water bath for 30 min, the drug-matrix mixture was placed into a disposable Ultrafree-MC (mol. wt. cutoff, 30,000) filtration system, which was centrifuged at 1500g for 30 min. An aliquot of protein-free filtrate and the initial samples were analyzed using a previously validated ELISA method (Wei et al., 2006b), which provides a low quantitation limit of 0.05 nM, and such sensitivity is required for measurement of GTI-2040 levels in ultrafiltrate. The unbound GTI-2040 fractions were estimated from the percentage of the concentrations in the filtrate compared with the initial concentrations. Samples prepared in saline were processed under the identical conditions and used as controls for nonspecific binding.

Similarly, human plasma protein binding was carried out at various total drug concentrations of 1, 10, and 100 μM in donor plasma. Following incubation and centrifugation with the Ultrafree-MC filtration system, an aliquot of protein-free filtrate and the initial samples were analyzed using the ELISA method. In addition, the in vivo free fraction of GTI-2040 in plasma (fu,p) for AML patients was also estimated by dividing the patient's renal clearance (CLR) with the glomerular filtration rate, which is estimated by patient's creatinine clearance (CrCL) (Rowland and Tozer, 1995): CLR for each patient was determined in eq. 8 (shown later).

Enzyme Kinetics of GTI-2040 in the Solution Containing 3′ Exonuclease. To prepare the 10 U/ml stock solution of phosphodiesterase I, the lyophilized enzyme was reconstituted in Tris-salt buffer/glycerol [50:50 (v/v)] containing 110 mM Tris-HCl, pH 8.9, 110 mM NaCl, and 15 mM MgCl2, and stored at -20°C before use. To conduct the enzyme kinetic study, various concentrations of GTI-2040 ranging from 0.1 to 20 μM were incubated with 0.3 U/ml phosphodiesterase I diluted in mobile phase A (100 mM HFIP titrated to pH 8.3 by 8.4 mM TEA) at 37°C for 0.5 h. This specific condition is required for the HPLC assay because the typically used buffer system was found to interfere with the separation of GTI-2040 and its metabolites. The reaction matrix has been shown previously to possess similar metabolic activity as in the buffer (Wei et al., 2006a). The incubation time and quantity of enzyme used were both within the linear region in the reaction condition. The reactions were terminated by the addition of 5 mM EDTA. Then, an appropriate amount of PS-dC 28 was added and used as the internal standard, and the samples were immediately frozen until analysis. For analysis, samples were thawed at 4°C and analyzed by HPLC without further preparation. Solutions spiked with the known initial levels of GTI-2040 in 0.3 U/ml phosphodiesterase I containing appropriate amounts of PS-dC 28 and 5 mM EDTA without enzyme reaction were prepared as GTI-2040 controls for the calculation of metabolite concentration.

Enzyme Kinetics of GTI-2040 in Pooled Human Liver Microsomes. Incubations of GTI-2040 were carried out under initial linear conditions with respect to time (30 min) and microsomal protein concentration (0.2 mg protein/ml). This protein concentration was selected based on the best linear condition from evaluation of 0.1, 0.2, and 0.5 mg/ml. Substrates at the concentration range between 0.1 and 20 μM were incubated with human liver microsomes in a 1-ml tube in a shaking water bath at 37°C with frequent inversed mixing. All incubations were performed in duplicate. At the end of incubation, 5 mM EDTA was added to stop the reaction. Appropriate amounts of the IS PS-dC 28 were added into each sample. The mixture was then centrifuged at 3000g for 2 min, and the supernatant was separated and stored at -80°C until analysis. GTI-2040 and its metabolites were extracted with solid phase extraction and analyzed by HPLC-UV. Another set of EDTA-treated HLM samples containing GTI-2040 at initial concentrations and PS-dC 28 without incubation was carried out with the extraction as described as above and was used as the control GTI-2040 for the calculation of the metabolite concentration.

Clinical Studies. A National Cancer Institute/Cancer Therapy Evaluation Program-sponsored phase I clinical trial of GTI-2040 in patients with refractory or relapsed AML was carried out at the Ohio State University James Cancer Hospital and Research Institute in adherence to the Institute Research Review Board. Informed consent was obtained before entry onto the study.

In Vivo Clearance. Patients with AML were treated with GTI-2040 at 3.5 or 5 mg/kg/day as a continuous i.v. infusion for a total of 144 h. Blood was drawn at various time points during and after infusion. Plasma was separated from the whole blood by centrifugation at 1400g for 20 min. Pretreatment and 24-h cumulative urine was also collected. GTI-2040 concentrations in plasma and urine were determined by the previously reported ELISA method (Dai et al., 2005a; Wei et al., 2006b).

Nonspecific Binding in HLMs. The nonspecific binding of GTI-2040 in HLMs is described by eq. 3. Kd and Bmax were calculated from the Scatchard equation (eq. 4), which is a linear transformation of eq. 3. where CB is the concentration of the bound drug in HLMs, CF is the free drug concentration in HLMs, Bmax is the maximal binding capacity, and Kd is the binding dissociation constant.

Calculation of the in Vitro CLint. The rate-substrate concentration profiles were fitted by the Michaelis-Menten equation (eq. 5) or Hill equation (eq. 6) using the nonlinear regression model in SigmaPlot (Systat Software, Inc., San Jose, CA). where v and Vmax are the observed and maximal rates of metabolism, Km is the Michaelis constant and is the concentration at half of Vmax in the Michaelis-Menten equation, S50 is the concentration at half of Vmax in the Hill equation,

n is the Hill coefficient, and S is the unbound substrate concentration. The in vitro CLint was then estimated from eq. 7:

Calculation of the in Vivo CLint,h from Patients. Renal clearance of GTI-2040 in patients was estimated by dividing the cumulative amount of GTI-2040 in 24-h urine with the 24-h plasma area under the curve, as shown in eq. 9. Assuming that the total clearance for GTI-2040 consists of mainly renal and hepatic clearances, and only minimal parent drugs would undergo biliary excretion (Lischda et al., 2003), the hepatic clearance from patients can be obtained, therefore, by subtracting renal clearance from the total plasma clearance (eq. 10). CLH is given by the following equation: which can be transformed into where fu,p is the free fraction of the drug in plasma and was determined from eq. 2. CLint,h is the in vivo intrinsic clearance estimated in patients. QH is the hepatic plasma flow rate and was estimated to be 49 l/h for a 70-kg human.

Scaling of the in Vitro CLint to the in Vivo CLint,h1. Microsomal protein amount was used as a scaling factor to transform the in vitro CLint in microsomes to the in vivo CLint,h1 value. Assuming that 1 g of liver contains approximately 50 mg of microsomal protein, and the liver of a 70-kg human weighs approximately 1400 g, a scaling factor (SF) of 70,000 mg is obtained (Kuhnz and Gieschen, 1998). Multiplication of the in vitro CLint by this scaling factor yields the scaled in vivo CLint,h1, expressed as liters per hour.

Results

Protein Binding. In human plasma, GTI-2040 was found to exhibit concentration-dependent high plasma protein binding. At total GTI-2040 concentrations of 0.5, 1, 10, and 100 μM, mean free drug fractions were found to be 0.05, 0.09, 2.2, and 5.5%, respectively (Table 1). When plasma total GTI-2040 concentration was <0.5 μM, free drug concentrations in the ultrafiltrate fell below the lower limit of quantitation (0.05 nM) of the ELISA assay. Because most of the clinically relevant plasma GTI-2040 concentrations are at about 0.5 μM (ranging from 0.114-0.728 μM), it is technically difficult to determine the free drug concentration by the ultrafiltration method. To obtain an assessment of the free fraction of drug in patients (fu,p), the in vivo fu,p for GTI-2040 was estimated by the ratio of renal clearance of patients to the corresponding creatinine clearance and was found to be in the range between 0.0034 and 0.256%, with a mean value of 0.071% in 21 patients. This result is in the same range as estimated by extrapolation from the in vitro protein binding as described above.

In HLMs, a concentration-dependent binding of GTI-2040 was observed as shown in Table 1 and Fig. 1. When GTI-2040 concentrations were increased from 0.1 to 20 μM, the free fraction of GTI-2040 in HLMs (0.2 mg protein/ml) was found to increase from 7.2 to 63.9%. As shown in Fig. 1A, the binding appears to be saturated when the substrate concentrations were higher than 10 μM. Two straight lines with different slopes were obtained from the Scatchard plots (Fig. 1B), indicating that more than one binding site was present with different affinities in HLMs for GTI-2040. A higher binding affinity with a binding dissociation constant Kd = 0.03 μM, a maximum binding capacity Bmax = 0.49 μM, and a lower binding affinity with Kd = 3.8 μM and Bmax = 8.9 μM were calculated from the Scatchard equation (eq. 4).

Kinetics of Metabolite Formation of GTI-2040 in 3′ Exonuclease Solution. Using the HPLC method, GTI-2040 and its 3′ chain-shortened metabolites derived from phosphodiesterase I were well separated (data not shown). At the incubation period of 0.5 h, 3′N-1 and 3′N-2 were found to be the two major metabolites in all evaluated substrate concentrations. The dose recoveries, calculated as the sum of percentage of parent and metabolites (3′N-1 and 3′N-2) to the added amounts of GTI-2040, were found to be in the range of 85.8 to 109.8% (mean, 98.8%) over the substrate level from 0.1 to 20 μM, indicating that the sum of 3′N-1 and 3′N-2 essentially accounts for the total metabolism of GTI-2040 during the initial incubation period.

Because metabolism of GTI-2040 was found mainly through sequential deletion of a single 3′ end nucleotide, enzyme kinetics of GTI-2040 in 0.3 U/ml phosphodiesterase I were evaluated using the sum of the formation rates of 3′N-1 and 3′N-2 metabolites (representing nearly total metabolism), and the results are presented in Fig. 2, A and B. As shown in Fig. 2A, a hyperbolic saturation curve was observed, and when transformed to the Eadie-Hofstee format, a linear plot was observed (Fig. 2B), conforming to the Michaelis-Menten kinetics. Therefore, the Michaelis-Menten equation was used to fit the formation kinetics of total metabolites. As determined by eq. 5, the Km was found to be 1.28 ± 0.42 μM, and the Vmax was 0.73 ± 0.06 nmol/h. CLint of GTI-2040 in 0.3 U/ml phosphodiesterase I, as the ratio of Vmax to Km, was calculated to be 0.60 ± 0.19 ml/h. The estimated kinetic constants are listed in Table 2.

A, velocity of the sum of formation rate of 3′N-1 and 3′N-2 versus GTI-2040 substrate total concentrations in HLMs (0.2 mg protein/ml). B, Eadie-Hofstee plot of the kinetics of the sum of formation rate of 3′N-1 and 3′N-2 in HLMs (0.2 mg protein/ml).

Kinetics of Metabolite Formation of GTI-2040 in Pooled Human Liver Microsomes. GTI-2040 and its metabolites from HLM incubation were well separated using the ion-pair HPLC system, and no interference was found from the control HLMs (data not shown). In the initial incubation period of 0.5 h, 3′N-1 and 3′N-2 were found to be the major metabolites in all of the tested substrate concentrations. The drug recoveries were estimated by comparison of the sum of the unchanged GTI-2040 and metabolites with the added amount of GTI-2040 and found to be in the range of 89.6 to 110.0%, with a mean value of 98.9% over the substrate concentrations from 0.1 to 20 μM, indicating that the sum of 3′N-1 and 3′N-2 essentially accounts for the total metabolism of GTI-2040 in HLMs during the initial incubation period.

The sum of the formation rates of the major metabolites 3′N-1 and 3′N-2 was plotted against the total substrate GTI-2040 concentrations, and a sigmoidal shape curve is shown in Fig. 3A. These data were reprocessed using an Eadie-Hofstee plot as shown in Fig. 3B. The Eadie-Hofstee plot exhibits a curvilinear curve instead of a commonly found straight line. A curved Eadie-Hofstee plot indicates deviation from the classical Michaelis-Menten model (Houston and Kenworthy, 2000; Houston and Galetin, 2005; Tracy, 2006). In this case, the Hill equation (eq. 6) is commonly used for estimation of the parameters for the compound displaying atypical kinetics, and the S50 was calculated to be 12.1 ± 8.6 μM, the Vmax 16.8 ± 8.0 nmol/h/mg protein, and the Hill coefficient 1.28 ± 0.4. The relevant kinetic constants as estimated are listed in Table 2. Because the metabolic system in HLM contains microsomal membrane, to examine the impact of the nonspecific binding of GTI-2040 in HLMs on the enzyme kinetic curves, the free fraction of GTI-2040 in HLMs was employed to calculate the formation rate of metabolites (Fig. 4A). As shown in Fig. 4B, the Eadie-Hofstee plots shows a linear trend using the free concentrations of substrate in the range from 0.0159 to 12.8 μM (corresponding to 0.1-20 μM of total drug concentrations). Metabolism kinetics of GTI-2040 in HLMs were fitted with a Michaelis-Menten model, and eq. 5 was used to calculate the kinetic rate constants. As listed in Table 2, after correcting with the free fraction of the substrate, the Km was estimated to be 6.33 ± 3.2 μM, the Vmax was 16.5 ± 8.4 nmol/h/mg protein, and the CLint (Vmax/Km) was 2.61 ± 0.56 ml/h.

Correlation of the in Vitro CLint from HLMs with the in Vivo CLint,h. Renal clearance from 21 AML patients was determined to be in the range between 0.00026 and 0.019 l/h, with a mean value of 0.0032 l/h. Following eqs. 9 to 11, the in vivo intrinsic clearance was found to vary from 238.7 to 2846.4 l/h, with a mean value at 758.7 l/h. Because it is the unbound drug that gains access to the active site of metabolic enzymes, the in vitro HLM CLint that calculated from the unbound GTI-2040 substrate concentrations was used to estimate the in vivo CLint,h1 using the microsomal protein scaling factor (eq. 12). In this fashion, the predicted in vivo CLint,h1 was determined to be 182.7 l/h, which represented 24.1% of the observed CLint,h in patients. Detailed clinical pharmacokinetics of GTI 2040 in AML patients have been published recently (Klisovic et al., 2008).

Discussion

In this study, enzyme kinetics of GTI-2040 were characterized as its formation rate of metabolites in both pure enzyme solution and human liver microsomes. In the 3′ exonuclease solution system, where protein binding is negligible, the formation rate of sum of 3′N-1 and 3′N-2, two main GTI-2040 metabolites, was well fitted with the Michaelis-Menten model (Fig. 2, A and B). Because the metabolism of GTI-2040 has been found to mainly undergo sequential nucleotide deletion via 3′ exonuclease, it is important to take into account its sequential metabolism to correctly interpret its enzyme kinetics. When only the 3′N-1 metabolite was considered as the sole pathway, the Eadie-Hofstee plot is nonlinear in the range of evaluated substrate concentrations, and the enzyme kinetics deviate from Michaelis-Menten model as a result of underestimation of the extent of metabolism (data not shown). However, when the sum of 3′N-1 and 3′N-2 metabolites was taken into account, the Eadie-Hofstee plot became linear, and the kinetics appeared to follow the Michaelis-Menten model. Because in this tested metabolism system, >85% dose of GTI-2040 was accounted for by the measurement of the sum of GTI-2040 and the 3′N-1 and 3′N-2 metabolites under the initial reaction condition, the use of the sum of 3′N-1 and 3′N-2 metabolites, therefore, should correctly describe the enzyme kinetics of GTI-2040 in the 3′ exonuclease solution.

A, velocity of sum of formation rate of 3′N-1 and 3′N-2 versus unbound GTI-2040 substrate concentrations. B, Eadie-Hofstee plot of the kinetics of the sum of formation rates of 3′N-1 and 3′N-2 in HLMs derived from unbound GTI-2040 (0.2 mg protein/ml).

When enzyme kinetics of GTI-2040 in HLMs were analyzed using the sum of the formation rate of 3′N-1 and 3′N-2, a sigmoidal kinetics of GTI-2040 was observed, and a curvilinear Eadie-Hofstee plot was obtained (Fig. 3, A and B). This would suggest an enzyme autoactivation by the substrate (Houston and Kenworthy, 2000; Tracy, 2006), and in such a case, a Hill equation may have to be used for enzyme kinetic analysis. However, after the formation rate of total metabolites was corrected using the free fractions of substrate GTI-2040 at each concentration level, an apparent, normal Michaelis-Menten plot but a biphasic Eadie-Hofstee plot composed of two linear segments approximately above and below an unbound substrate concentration of about 0.112 μM (equivalent to total substrate concentration at 0.5 μM) was shown (Fig. 4B). This biphasic Eadie-Hofstee plot might relate to our previous Scatchard analysis (Fig. 1; Table 1), which shows two binding sites with rather different binding dissociate constants because the slope of the Eadie-Hofstee plot reflects the Km of GTI-2040 to the HLMs. This renders the enzyme kinetics analysis rather complex in HLMs, if both binding sites are included. For example, if the fraction of unbound drug in HLMs [fu(mic)] is constant over the range of substrate concentrations, the apparent Km,app value is usually converted to the free Km by multiplication of fu(mic) (Tang et al., 2002; Isoherranen et al., 2004). However, if the binding is concentration dependent, and the substrate concentration range used (the determined Km value as well) is similar to or higher than the Kd, fractions of the unbound drug [fu(mic)] may vary over the substrate concentration range used, and free Km value cannot be calculated from a constant fu(mic). Then, in such a case, sigmoidal kinetics and curvilinear Eadie-Hofstee plots could arise as a consequence to the various free fractions of the substrate available to the enzyme (Houston and Kenworthy, 2000; McLure et al., 2000). Thus, the sigmoidal kinetics of GTI-2040 observed for HLMs may be because of, in part, a concentration-dependent binding to microsomal membrane.

Because it is the unbound drugs that become available to interact with enzyme, the unbound substrate concentrations mostly fell below the binding dissociation constants to the HLMs and the Km for the 3′-exonuclease of 1.28 μM, we elected to approximate the enzyme kinetics in the HLMs using the Michaelis-Menten model. Additionally, clinically GTI-2040 is used as a 6-day infusion, and the total steady-state concentrations are in the range of 0.3 μM (Klisovic et al., 2008), further justifying the use of the apparent Michaelis-Menten kinetics to describe the GTI-2040 data for approximation of the HLM enzyme kinetics, which yielded the kinetic parameter as shown in Table 2. Notably, the Km value was determined to be 6.33 μM, using the unbound substrate concentrations, and this value is greater than both Kd values of GTI-2040 in HLMs. This approximation is especially relevant clinically because the steady-state drug concentrations seen in circulation were well above 0.1 μM (Klisovic et al., 2008) and would be adequate to estimate the in vitro intrinsic clearance of GTI-2040 in HLMs, which allows for the in vitro-in vivo correlation.

In vivo intrinsic clearance from patients was compared with the predicted CLint,h1 that was scaled up from the CLint in HLMs. The predicted in vivo CLint,h1 accounted for 24.1% of the observed in vivo CLint,h. This might indicate that human liver microsomes only partially contributed to the metabolism of GTI-2040. The underestimation of the in vivo CLint,h1 is likely because of the multiple metabolism sites of PS-ODNs in vivo. In vivo, PS-ODNs extensively distribute to different organs and tissues with the majority of drugs accumulating within the liver and the kidneys (Agrawal et al., 1991; Griffey et al., 1997; Yu et al., 2004; Noll et al., 2005). In the liver, PS-ODNs are not only taken up into the hepatocytes but also rapidly distribute to the nonparenchymal cells, including Kupffer cells and endothelial cells (Nolting et al., 1997; Graham et al., 2001), and only a small amount of drug is excreted into the bile (Lischka et al., 2003). In addition to the liver, kidneys are another important organ responsible for the degradation of PS-ODN. Significant amounts of chain-shortened metabolites were detected in the kidneys, especially in the proximal tubular cells (Griffey et al., 1997; Noll et al., 2005). Metabolism of GTI-2040 and other PS-ODNs was also found in circulation and other organ tissues in addition to liver (Gilar et al., 1997; Wei et al., 2006a). Therefore, liver microsomes may not be the sole responsible system for the full prediction of the in vivo metabolism of GTI-2040. In addition, it is reported that in vivo human intrinsic clearance is usually underpredicted using human liver microsomes, possibly because of the variability of the source of liver donated (Brown et al., 2007). The current predicted value is considered not unreasonable relative to other drugs.

This is the first time that an enzyme kinetics study of GTI-2040 was investigated in a pure enzyme solution and in human liver microsomes. It is interesting to observe sigmoidal kinetics of GTI-2040 in HLMs, which we attributed to the complex effects such as the sequential metabolism and the saturable nonspecific binding in HLMs. Whether this phenomenon applies to other PS-ODNs remains to be demonstrated. Nevertheless, our studies suggest that careful interpretation of the enzyme kinetics of PS-ODNs, with respect to the presence of sequential metabolism, high protein binding, and the saturable nonspecific binding in the metabolism systems, should be made. In addition, we have found that the scaled intrinsic clearance from the in vitro HLMs represented 24.1% of GTI-2040 in vivo intrinsic clearance from AML patients probably is because of the partial contribution of liver microsomes in metabolism of GTI-2040.

Footnotes

This study was supported by the National Institutes of Health (Grant NCI R21 CA 105879; to G.M.).

Brown HS, Griffin M, and Houston JB (2007) Evaluation of cryopreserved human hepatocytes as an alternative in vitro system to microsomes for the prediction of metabolic clearance. Drug Metab Dispos35:293-301.